Ilia N. Buhtoiarov

Enhanced activity of hu14.18-IL2 immunocytokine against murine NXS2 neuroblastoma when combined with interleukin 2 therapy.

Established s.c. NXS2 murine neuroblastoma tumors exhibited transient resolution after suboptimal therapy using the hu14.18-IL2 immunocytokine (IC). The hu14.18-IL2 IC is a fusion protein that has linked a molecule of interleukin 2 (IL-2) to the COOH terminus of each of the IgG heavy chains on the humanized anti-GD2 monoclonal antibody hu14.18. To induce more potent and longer lasting in vivo antitumor effects, we tested hu14.18-IL2 IC in a regimen combining it with constant infusion IL-2 in NXS2 tumor-bearing mice. The addition of the constant infusion IL-2 augmented the antitumor response induced by treatment with the hu14.18-IL2 IC in animals with experimentally induced hepatic metastases and in animals bearing localized s.c. tumors. The combined treatment induced prolonged tumor eradication in most animals bearing s.c. tumors and involved both natural killer cells and T cells. The enhanced ability of this combined treatment to prevent tumor recurrence was not observed when a larger dose of hu14.18-IL2 IC, similar in IL-2 content to the IC plus systemic IL-2 regimen, was tested as single-agent therapy. Animals showing prolonged tumor eradication of established tumors after the combined hu14.18-IL2 plus IL-2 regimen exhibited a protective T-cell-dependent antitumor memory response against NXS2 rechallenge.

CD40 Ligation Activates Murine Macrophages via an IFN-γ-Dependent Mechanism Resulting in Tumor Cell Destruction In Vitro

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-γ because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-γ mAb, as well as in IFN-γ-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-γ for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-γ. We confirmed this hypothesis by detecting intracytoplasmic IFN-γ in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-γ production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-γ and mediate tumor cell destruction.

Synergistic Activation of Macrophages via CD40 and TLR9 Results in T Cell Independent Antitumor Effects

We have previously shown that macrophages (Mφ) can be activated by CD40 ligation to become cytotoxic against tumor cells in vitro. Here we show that treatment of mice with agonistic anti-CD40 mAb (anti-CD40) induced up-regulation of intracellular TLR9 in Mφ and primed them to respond to CpG-containing oligodeoxynucleotides (CpG), resulting in synergistic activation. The synergy between anti-CD40 and CpG was evidenced by increased production of IFN-γ, IL-12, TNF-α, and NO by Mφ, as well as by augmented apoptogenic effects of Mφ against tumor cells in vitro. The activation of cytotoxic Mφ after anti-CD40 plus CpG treatment was dependent on IFN-γ but not TNF-α or NO, and did not require T cells and NK cells. Anti-CD40 and CpG also synergized in vivo in retardation of tumor growth in both immunocompetent and immunodeficient mice. Inactivation of Mφ in SCID/beige mice by silica treatment abrogated the antitumor effect. Taken together, our results show that Mφ can be activated via CD40/TLR9 ligation to kill tumor cells in vitro and inhibit tumor growth in vivo even in immunocompromised tumor-bearing hosts, indicating that this Mφ-based immunotherapeutic strategy may be appropriate for clinical testing.

Anti-tumour synergy of cytotoxic chemotherapy and anti-CD40 plus CpG-ODN immunotherapy through repolarization of tumour-associated macrophages.

We studied the effectiveness of monoclonal anti‐CD40 + cytosine–phosphate–guanosine‐containing oligodeoxynucleotide 1826 (CpG‐ODN) immunotherapy (IT) in mice treated with multidrug chemotherapy (CT) consisting of vincristine, cyclophosphamide and doxorubicin. Combining CT with IT led to synergistic anti‐tumour effects in C57BL/6 mice with established B16 melanoma or 9464D neuroblastoma. CT suppressed the functions of T cells and natural killer (NK) cells, but primed naïve peritoneal macrophages (Mφ) to in vitro stimulation with lipopolysaccharide (LPS), resulting in augmented nitric oxide (NO) production. IT, given after CT, did not restore the responsiveness of T cells and NK cells, but further activated Mφ to secrete NO, interferon‐γ (IFN‐γ) and interleukin (IL)‐12p40 and to suppress the proliferation of tumour cells in vitro. These functional changes were accompanied by immunophenotype alterations on Mφ, including the up‐regulation of Gr‐1. CD11b+ F4/80+ Mφ comprised the major population of B16 tumour‐infiltrating leucocytes. CT + IT treatment up‐regulated molecules associated with the M1 effector Mφ phenotype [CD40, CD80, CD86, major histocompatibility complex (MHC) class II, IFN‐γ, tumour necrosis factor‐α (TNF‐α) and IL‐12] and down‐regulated molecules associated with the M2 inhibitory Mφ phenotype (IL‐4Rα, B7‐H1, IL‐4 and IL‐10) on the tumour‐associated Mφ compared with untreated controls. Together, the results show that CT and anti‐CD40 + CpG‐ODN IT synergize in the induction of anti‐tumour effects which are associated with the phenotypic repolarization of tumour‐associated Mφ.

In vivo CD40 ligation can induce T cell‐independent antitumor effects that involve macrophages

We have previously demonstrated T cell‐independent antitumor and antimetastatic effects of CD40 ligation that involved natural killer (NK) cells. As CD40 molecules are expressed on the surface of macrophages (Mφ), we hypothesized that Mφ may also serve as antitumor effector cells when activated by CD40 ligation. Progression of subcutaneous NXS2 murine neuroblastomas was delayed significantly by agonistic CD40 monoclonal antibody (anti‐CD40 mAb) therapy in immunocompetent A/J mice, as well as in T and B cell‐deficient severe combined immunodeficiency (SCID) mice. Although NK cells can be activated by anti‐CD40 mAb, anti‐CD40 mAb treatment also induced a significant antitumor effect in SCID/beige mice in the absence of T and NK effector cells, even when noncytolytic NK cells and polymorphonuclear cells (PMN) were depleted. Furthermore, in vivo treatment with anti‐CD40 mAb resulted in enhanced expression of cytokines and cell surface activation markers, as well as Mφ‐mediated tumor inhibition in A/J mice, C57BL/6 mice, and SCID/beige mice, as measured in vitro. A role for Mφ was shown by reduction in the antitumor effect of anti‐CD40 mAb when Mφ functions were inhibited in vivo by silica. In addition, activation of peritoneal Mφ by anti‐CD40 mAb resulted in survival benefits in mice bearing intraperitoneal tumors. Taken together, our results show that anti‐CD40 mAb immunotherapy of mice can inhibit tumor growth in the absence of T cells, NK cells, and PMN through the involvement of activated Mφ.

Intratumoral immunocytokine treatment results in enhanced antitumor effects

Immunocytokines (IC), consisting of tumor-specific monoclonal antibodies fused to the immunostimulatory cytokine interleukin 2 (IL2), exert significant antitumor effects in several murine tumor models. We investigated whether intratumoral (IT) administration of IC provided enhanced antitumor effects against subcutaneous tumors. Three unique ICs (huKS-IL2, hu14.18-IL2, and GcT84.66-IL2) were administered systemically or IT to evaluate their antitumor effects against tumors expressing the appropriate IC-targeted tumor antigens. The effect of IT injection of the primary tumor on a distant tumor was also evaluated. Here, we show that IT injection of IC resulted in enhanced antitumor effects against B16-KSA melanoma, NXS2 neuroblastoma, and human M21 melanoma xenografts when compared to intravenous (IV) IC injection. Resolution of both primary and distant subcutaneous tumors and a tumor-specific memory response were demonstrated following IT treatment in immunocompetent mice bearing NXS2 tumors. The IT effect of huKS-IL2 IC was antigen-specific, enhanced compared to IL2 alone, and dose-dependent. Hu14.18-IL2 also showed greater IT effects than IL2 alone. The antitumor effect of IT IC did not always require T cells since IT IC induced antitumor effects against tumors in both SCID and nude mice. Localization studies using radiolabeled 111In-GcT84.66-IL2 IC confirmed that IT injection resulted in a higher concentration of IC at the tumor site than IV administration. In conclusion, we suggest that IT IC is more effective than IV administration against palpable tumors. Further testing is required to determine how to potentially incorporate IT administration of IC into an antitumor regimen that optimizes local and systemic anticancer therapy.

Macrophages are essential for antitumour effects against weakly immunogenic murine tumours induced by class B CpG-oligodeoxynucleotides.

We explored the mechanisms of class B CpG‐oligodeoxynucleotide‐induced antitumour effects against weakly immunogenic tumours. Treatment with CpG‐oligodeoxynucleotide 1826 (CpG) induced similar antitumour effects in B16 melanoma‐bearing immunocompetent C57BL/6 mice and T‐cell‐deficient severe combined immunodeficient (SCID) mice, and NXS2 neuroblastoma‐bearing T‐cell‐depleted A/J mice. Both macrophages (Mφ) and natural killer (NK) cells from CpG‐treated C57BL/6 mice could mediate cytotoxicity in vitro, suggesting that these cell types might control tumour growth in vivo. However, CpG treatment of SCID/beige mice or T‐cell‐depleted and NK‐cell‐depleted A/J mice still induced antitumour effects in vivo, arguing against a major role of NK cells in the antitumour effects of CpG in the absence of T cells. In contrast, CpG treatment of interferon‐γ knockout (IFN‐γ–/–) C57BL/6 mice resulted in no antitumour effects in vivo and no Mφ‐mediated tumoristasis in vitro despite unaltered cytolytic function of NK cells in vitro. Moreover, Mφ inactivation by silica substantially reduced CpG‐induced suppression of tumour growth in vivo, revealing an important role of Mφ in CpG‐induced antitumour effects. The in vitro tumouritoxicity by CpG‐stimulated Mφ (CpG‐Mφ) correlated with tumour cell mitochondria dysfunction and involved nitric oxide (NO), tumour necrosis factor‐α (TNF‐α) and IFN‐γ, whereas interleukin‐1α (IL‐1α), IL‐1β, IFN‐α, TNF‐related apoptosis‐inducing ligand and Fas ligand played insignificant roles in CpG‐Mφ tumouritoxicity. Taken together, our results indicate that the growth control of weakly immunogenic tumours during CpG‐immunotherapy is mediated predominantly by Mφ, rather than T cells or NK cells.

Tumoristatic effects of anti-CD40 mAb-activated macrophages involve nitric oxide and tumour necrosis factor-α

Effector cells of the innate immune system have diverse functions that can result in tumour inhibition or tumour progression. Activation of macrophages by CD40 ligation has been shown to induce antitumour effects in vitro and in vivo. Here we investigated the role of nitric oxide (NO) and tumour necrosis factor‐α (TNF‐α) as mediators in the tumoristatic effects of murine peritoneal macrophages activated with agonistic anti‐CD40 monoclonal antibody (αCD40) alone and following further stimulation with bacterial lipopolysaccharide (LPS). We found that macrophages activated in vivo by αCD40 exhibited tumoristatic activity in vitro against B16 melanoma cells; the tumoristatic effect correlated with the level of NO production and was enhanced by LPS. Use of the NO inhibitor l‐nitro‐arginine‐methyl esterase (L‐NAME) and evaluation of macrophages from inducible NO synthase (iNOS)‐knockout (KO) mice following αCD40 activation showed reduced tumoristatic activity. CD40 ligation enhanced expression of TNF‐α. Macrophage tumoristatic activity following αCD40 treatment was reduced by TNF‐α mAb or use of macrophages from TNF‐α‐KO mice. However, further stimulation of αCD40‐activated macrophages with LPS resulted in strong tumoristatic activity that was much less dependent on NO or TNF‐α. Taken together, these results suggest that NO and TNF‐α are involved in, but not solely responsible for, the antitumour effects of macrophages after activation by CD40 ligation.

Ab-IL2 fusion proteins mediate NK cell immune synapse formation by polarizing CD25 to the target cell-effector cell interface

The huKS-IL2 immunocytokine (IC) consists of IL2 fused to a mAb against EpCAM, while the hu14.18-IL2 IC recognizes the GD2 disialoganglioside. They are under evaluation for treatment of EpCAM+ (ovarian) and GD2+ (neuroblastoma and melanoma) malignancies because of their proven ability to enhance tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and by antitumor cytotoxic T cells. Here, we demonstrate that huKS-IL2 and hu14.18-IL2 bind to tumor cells via their antibody components and increase adhesion and activating immune synapse (AIS) formation with NK cells by engaging the immune cells’ IL-2 receptors (IL2R). The NK leukemia cell line, NKL (which expresses high affinity IL2Rs), shows fivefold increase in binding to tumor targets when treated with IC compared to matching controls. This increase in binding is effectively inhibited by blocking antibodies against CD25, the α-chain of the IL2R. NK cells isolated from the peritoneal environment of ovarian cancer patients, known to be impaired in mediating ADCC, bind to huKS-IL2 via CD25. The increased binding between tumor and effector cells via ICs is due to the formation of AIS that are characterized by the simultaneous polarization of LFA-1, CD2 and F-actin at the cellular interface. AIS formation of peritoneal NK and NKL cells is inhibited by anti-CD25 blocking antibody and is 50–200% higher with IC versus the parent antibody. These findings demonstrate that the IL-2 component of the IC allows IL2Rs to function not only as receptors for this cytokine but also as facilitators of peritoneal NK cell binding to IC-coated tumor cells.

Radiofrequency ablation combined with KS-IL2 immunocytokine (EMD 273066) results in an enhanced antitumor effect against murine colon adenocarcinoma.

Purpose: Radiofrequency ablation (RFA) is a common treatment modality for surgically unresectable tumors. However, there is a high rate of both local and systemic recurrence. Experimental Design: In this preclinical study, we sought to enhance the antitumor effect of RFA by combining it with huKS-IL2 immunocytokine [tumor-specific monoclonal antibody fused to interleukin-2 (IL2)] in mice bearing CT26-KS colon adenocarcinoma. Mice were treated with RFA, huKS-IL2 via intratumoral injection, or combination therapy. Results: Treatment of mice bearing s.c. tumors with RFA and huKS-IL2 resulted in significantly greater tumor growth suppression and enhanced survival compared with mice treated with RFA or huKS-IL2 alone. When subtherapeutic regimens of RFA or huKS-IL2 were used, tumors progressed in all treated mice. In contrast, the combination of RFA and immunocytokine resulted in complete tumor resolution in 50% of mice. Treatment of a tumor with RFA and intratumoral huKS-IL2 also showed antitumor effects against a distant untreated tumor. Tumor-free mice after treatment with RFA and huKS-IL2 showed immunologic memory based on their ability to reject subsequent challenges of CT26-KS and the more aggressive parental CT26 tumors. Flow cytometry analysis of tumor-reactive T cells from mice with complete tumor resolution showed that treatment with RFA and huKS-IL2 resulted in a greater proportion of cytokine-producing CD4 T cells and CD8 T cells compared with mice treated with RFA or huKS-IL2 alone. Conclusions: These results show that the addition of huKS-IL2 to RFA significantly enhances the antitumor response in this murine model, resulting in complete tumor resolution and induction of immunologic memory.